1. Field of the Invention
The present invention relates generally to improved methods for identifying and treating individuals who suffer from or are susceptible to diabetes. More particularly, the present invention relates to the use of glutamic acid decarboxylase to detect pancreatic .beta.-cell specific autoantibodies in sera of diabetic or prediabetic individuals and inhibit the immune response which causes .beta.-cell destruction.
Insulin dependent diabetes mellitus (IDDM) primarily afflicts young people. Although insulin is available for treatment, the several fold increased morbidity and mortality associated with this disease urge the development of early diagnostic and preventive methods. The destruction of pancreatic .beta.-cells, which precedes the clinical onset of IDDM, is mediated by autoimmune mechanisms. Among the most thoroughly studied autoimmune abnormalities associated with the disease is the high incidence of circulating .beta.-cell specific autoantibodies at the time of diagnosis. Family studies have shown that the autoantibodies appear prior to overt IDDM by a number of years, suggesting a long prodromal period of humoral autoimmunity before clinical symptoms emerge. The family studies have also documented a slow, progressive loss of insulin response to intravenous glucose in the years preceding diagnosis. The presence of .beta.-cell specific autoantibodies in the prediabetic period is likely to reflect the ongoing autoimmune process, one that eventually leads to critical .beta.-cell depletion and insulin dependency. It has been estimated that only 10% of the total .beta.-cell mass remains at the time of clinical onset.
A major goal of diabetes research has been to develop immune interventions that block or inhibit the destruction of .beta.-cells and development of IDDM. Since immune interventions will probably always confer some risk of untoward side effects, highly sensitive, specific and easily applicable markers for the prediabetic stage are needed if such a treatment is to become an acceptable alternative to replacement therapy with insulin.
In addition to preventative treatment, methods for early and accurate identification of susceptible individuals are needed. Assays that can detect autoantibodies associated with early humoral autoimmunity accompanying .beta.-cell destruction are particularly desirable. The classical method for detecting islet cell autoantibodies is by immunohistology using frozen pancreatic sections. Family studies, however, have shown that the .beta.-cell cytoplasmic antibodies (ICCA) measured by this method are of insufficient specificity to serve as a single marker of susceptibility. Moreover, ICCA are very difficult to standardize and interpretation of the stained section is subject to observer bias. Thus, there has been no way to define what is a "positive" specimen. More accurate assays may be achieved by employing more specific markers, either alone or in combination with ICCA. Alternative markers include 64k autoantibodies, insulin autoantibodies, and MHC class II DR/DQ.beta. haplotype.
Assays for the early detection of humoral responses in IDDM should provide a rapid and quantitative measurement of the particular marker, preferably by conventional serum assay protocols, such as enzyme linked immunosorbent assay (ELISA) and/or a radioimmunoassay (RIA). Such assays should detect a primary .beta.-cell target antigen characteristic of humoral autoimmunity and should have improved sensitivity and specificity relative to the ICCA assay in predicting IDDM. Insulin and the 64 k .beta.-cell autoantigen are currently the only known potential primary target antigens of humoral autoimmunity in IDDM, if .beta.-cell expression and specificity are applied as criteria for such an antigen. Insulin autoantibodies have an incidence of only 30-40% in young children, and much lower in older children and adults, who develop IDDM. The 64 k autoantibodies have an incidence of about 80% both at the time of clinical onset and in the prediabetic period and have been shown to precede overt IDDM by several years in familial studies and to be detected concomitantly with and sometimes before, but never later than ICCA and IAA. Thus, the detection of 64 k autoantibodies promises to be a useful approach for early detection of IDDM.
Detection of the 64 kD autoantigen, however, has been problematic. The 64 kD autoantigen was heretofore only identified in the pancreatic .beta.-cell, and has not been purified in sufficient quantities to allow sequencing, cloning, or other identification which would have permitted large scale preparation of reagents necessary for detection or therapy. For these reasons, detection of the 64 k autoantibody has been by immunoprecipitation with detergent lysates of rat and human islets. Such assays are expensive, time consuming and not easily adapted to clinical laboratory settings. Therefore, they would not be suitable for widespread screening of human populations to identify individuals at risk for developing IDDM.
For these reasons, it would be desirable to identify the nature of the 64 kD autoantigen so that large quantities of the autoantigen or analogs thereof could be obtained for use as reagents in detection and therapy of IDDM.
2. Description of the Relevant Art
The 64 kD autoantigen in pancreatic .beta.-cells was identified originally as a target of autoantibodies in IDDM by immunoprecipitation experiments using detergent lysates of human islets (Baekkeskov et al. (1982) Nature 298:167-169). Antibodies to the 64 kD autoantigen precede the clinical onset of IDDM and have been shown to have an incidence of about 80% at clinical onset and during the prediabetic period (Baekkeskov et al. (1987) J. Clin. Invest. 79:926-934; Atkinson et al. (1990) Lancet 335:1357-1360; and Christie et al. (1988) Diabetologia 31:597-602). The rat and human 64 kD protein are highly homologous with regard to autoantigenic epitopes (Christie et al. (1990) J. Biol. Chem. 265:376-381). The 64 kD autoantigen in islets of Langerhans is detected in three different forms with regard to hydrophobicity and compartmentalization: a hydrophilic soluble form of 65 kD and pI of approximately 7.1; a 64 kD hydrophobic form, which is soluble or of a low membrane avidity and has a pI of approximately 6.7; and a hydrophobic firmly membrane anchored form of the same electrophoretic mobility and pI. Both the membrane bound and the soluble hydrophobic 64 kD forms exist as two isoforms, .alpha. and .beta., which have identical pI and hydrophobic properties but differ by approximately 1 kD (Baekkeskov et al. (1989) Diabetes 38:1133-1141). The 64 kD autoantigen was found to be .beta.-cell specific in an analysis of a number of tissues, which did not include the brain (Christie et al., supra.).
Patients with a rare but severe neurological disease, Stiff Man Syndrome (SMS), have autoantibodies to GABA-ergic neurons. Glutamic acid decarboxylase (GAD), the enzyme that synthesizes GABA from glutamic acid, was found to be the predominant autoantigen (Solimena et al. (1988) N. Engl. J. Med. 318:1012-1020 and Solimena et al. (1990) N. Engl. J. Med. 322:1555-1560). Almost all the GABA-ergic neuron autoantibody positive patients were also positive for islet cell cytoplasmic antibodies, as demonstrated by immunofluorescence of pancreatic sections, and one third had IDDM. In addition, autoantibodies to GABA-ergic neurons, detectable by immunocytochemistry, were detected in 3 of 74 IDDM patients without SMS (Solimena et al. (1988) supra. and Solimena et al. (1990) supra.). Other studies have also reported a high incidence of IDDM in SMS patients (Lorish et al. (1989) Mayo Clin. Proc. 64:629-636). GAD is found at high levels in islets of Langerhans (Okada et al. (1976) Science 194:620-622). Within the islet, GAD is selectively localized to .beta.-cells and absent in the other three endocrine cell types, the .alpha., .delta., and PP cells (Garry et al. (1988) J. Histochem. & Cytochem. 36:573-580 and Vincent (1983) Neuroendocrinol. 36:197-204). While little is known about the biochemical properties of pancreatic GAD, the brain protein has been partially characterized. In the rat brain, 60% of the protein was found to be membrane bound and 40% was soluble following homogenization (Chang and Gottlieb (1988) J. Neurosci. 8:2123-2130). GAD in brain consists of at least two isomers resolved by differences in electrophoretic mobility in SDS-PAGE. Their molecular weights have been described as 59-66 kD (Chang and Gottlieb, supra.). Immunogenic epitopes of both isoforms are highly conserved from rodents to humans (Legay et al. (1986) J. Neurochem. 46:1478-1486). The mRNA of the larger form has been cloned and sequenced (Kaufman et al. (1986) Science 232:1138-1140 and Julien et al. (1990) J. Neurochem. 54:703-705. The protein is detected in a dimer form under non-reducing conditions (Legay (1987) J. Neurochem. 48:1022-1026). Erlander et al. (1991) Neuron 7:91-100 describes the cloning and sequencing of both the higher and lower molecular weight forms of rat CNS GAD. Cram et al. (1991) describes the partial sequencing of the brain and pancreatic forms of human GAD and reports seven amino acid substitutions over a 180 amino acid sequence. The sequence of the gene encoding the higher molecular weight form of rat CNS GAD is reported in Julien et al. (1990) J. Neurochem. 54:703 705 and Wyborski et al. (1990) Mol. Brain Res. 8:193-198. Portions of the experimental work underlying the present invention were reported in Baekkeskov et al. (1990) Nature 347:151-156.